With the advent of genetic engineering, it has become a major goal to modify and improve plants by introducing foreign genes encoding important functional traits. Such traits might include resistance to herbicides, pesticides, or pests; tolerance to cold, heat, drought, or salinity; or improved nutritional quality or yield of specific plant products. The current population explosion and concomitant world food and fiber shortage demand improved productivity in agricultural efforts since virtually all of the readily available, relatively fertile cropland in developed countries has already been placed in use [Science 214: 1087-1089 (1981)]. Modification of monocotyledonous plants including the cereals and many food crops would provide major nutritional and economic benefits.
Two approaches currently exist for transferring heterologous gene(s) or gene sequence(s) into the genome of plants, also known as plant transformation. The first, as described by Chilton et al. [Cell 11: 263-271, (1977)],relies on infection by Agrobacterium bacteria, which inserts sequences of a plasmid, known as the Ti-plasmid, into the genome of plant cells. Another approach, described by Lorz et al. [Mol. Genet., 199: 178-182 (1985)], known as direct transformation, induces uptake and integration of plasmid or linearized DNA into the genome of plant protoplasts (which are single cells stripped of cell wall material). The second approach may also use the Ti-plasmid, and has been shown to be efficacious. It is described by Krens et al. [Nature, 296: 72-74 (1982)].
Aqrobacterium mediated genetic transfers result from the activity of virulence genes encoded by the Ti-plasmid. The virulence genes effect integration of a region on the Ti-plasmid, designated T-DNA, into the plant genome. When the T-DNA region of the pathogen is manipulated to contain genes of interest, such genes will be integrated into the plant genome following infection. Whole plants, plant tissues, plant cells, and plant protoplasts can all be infected with Aqrobacterium.
Alternatively, integration of exogenous genes and gene sequences into plant genomes can be achieved by inducing DNA uptake into plant protoplasts. When protoplasts and DNA molecules are incubated together, under proper inducing conditions (i.e. the use of polyethylene glycol, liposomes and/or electroporation), DNA is taken up and integrated into the plant genome. The frequency of transformation is highly variable, however, and very few major crop plants can be regenerated from protoplasts.
Useful Aqrobacterium mediated transformation of dicot tissues and cells is limited, because many of the tissue cultures fail to regenerate plants. For leaf disk transformation, the Aqrobacterium Ti-plasmid is modified so that the phytohormone synthesis genes are deleted and so that the T-DNA includes gene(s) of interest. The modified Aqrobacterium is incubated with leaf disk explants in tissue culture. Following infection, the bacteria are killed with an antibiotic. Cells of the leaf disks are then induced to regenerate into plants through phytohormone manipulations. Presence of the T-DNA sequence can then be scored if an antibiotic resistance gene had been incorporated as a selective marker. The procedure has proven to be effective for transformation of model systems, i.e. tobacco, petunia, and tomato as described by Horsch et al. [Science, 227: 1229-1231 (1985) ]. However, cells of the leaf disks from many major crop plants, i.e. soybean, corn, wheat, sunflower, etc., will not regenerate into intact plants. The same regeneration problem is encountered in other Aqrobacterium infection systems including other plant tissues, cells, and protoplasts. Therefore, transfer of a gene(s) or gene sequence(s) into cell cultures has no significant impact on major crop plant improvement at this time.
Aqrobacterium mediated plant transformation has been less successful in monocots than dicots. Integration of T-DNA has been demonstrated in only a few non-regenerable monocot systems, namely, Chlorophytum and Narcissus as demonstrated by Hooykaas Van Slogteren et al. [Nature 311: 763-764 (1984)] and Lolium as described by Potrykus et al. [Mol. Gen. Genet. 199: 183-188 (1985)]. At the present time this approach is not considered useful in transformation of major monocot crops, i.e. corn, wheat, rice, etc.
Direct transformation through uptake of plasmid or linearized plasmid DNA by protoplasts is also limited by the requirement to regenerate plants from the protoplasts. None of the major crop plants, i.e. corn, wheat, barley, can be regenerated from protoplasts at frequencies which make the technique acceptable.
Genetic transformation of major crop plants therefore presents a significant problem. The possibility that exogenous DNA might be transferred into germinating pollen grains to modify plant properties has been considered. As the pollen tube emerges from the mature pollen grain, cell wall material is deposited behind the growing tip. Therefore, immediately behind the growing point, the cell wall is just beginning to form. Exogenous DNA may be able to enter the male gametophyte, and be carried to the egg during the course of pollen tube growth and fertilization.
A series of papers by Dieter Hess between 1976 and 1980 reported plant transformation through uptake of intact bacteriophages into germinating pollen [(Hess et al., Z. Pflanzinphysiol., 74: 371-376 (1974)]. In another publication, Hess et al. [Z. Pflanzinphysiol., 77: 247-254 (1976)] describe that Nicotiana glauca pollen was incubated with N. langsdorffii DNA and that progeny from the treated pollen showed heritable increased tumor formation as normally seen in the sexual cross. Also, Hess [Z. Pflanzinphysiol., 90: 119-132 (1978)] described experiments in which Petunia hybrida pollen was treated with lac-transducing phages. Progeny derived from the treated pollen showed improved growth on lactose media. Similar results were obtained by Hess [Z. Pflanzinphysiol., 93: 429-436 (1979)] by treating Petunia pollen with gal transducing phages. He also showed that when pollen from a pure line of Petunia hybrida with white flowers was incubated with DNA from red flowered Petunia lines, progeny derived from treated pollen subsequently expressed anthocyanin in the floral parts [Z. Pflanzinphysiol., 98: 321-337 (1980)]. The significance of these studies is not clear, however, since the gene responsible for anthocyanin synthesis has been shown to be hypervariable in embryonic cells and reversion to full expression has been observed at the same frequency as a result of somatic mutations.
PCT Patent Application WO 85/01856 discloses a method for transferring genes between maize inbreds using pollen as a vector comprising the steps of (a) obtaining DNA from a selected donor plant and optionally placing said DNA in a buffer and/or storing it; (b) removing mature pollen from a chosen pollen-donor plant; (c) germinating the pollen; (d) incubating the germinated pollen with the donor DNA; (e) pollinating the pollen-donor plant or other compatible mother plants with the treated pollen; (f) harvesting the resultant seed from the plant; (g) germinating the seed and screening for transformed plants. The method is quite inefficient as demonstrated for maize, however, in that the majority of ears receiving DNA treated pollen produced no caryopses and only 1 to 5 well developed caryopses developed per inflorescence pollinated in those ears which set seed. The maximum number of caryopses produced was 50 per influorescence, compared to between 300 and 500 caryopses following pollination with untreated pollen. In addition, only about 24 percent of the caryopses resulting from the DNA-treated pollen germinated while about 91 percent of untreated caryopses germinated.
Y. Ohta [Proc. Natl. Acad. Sci. U.S.A., 83: 715-719 (1986)] discloses a method for transformation of Zea mays Linnaeus. Plants were self-pollinated with pollen which had been incubated with DNA prepared from plant leaves of a corn strain carrying dominant alleles for a set of markers for which the recipient has recessive alleles. The high molecular weight DNA was suspended in 0.3M sucrose at a concentration of 40 ug (u=micro) per mL, and added to fresh pollen from a recipient plant to make a pasty DNA/pollen mixture. The mixture was then placed on the silks of the recipient plant for self-pollination. Maize plants pollinated with the DNA/pollen mixture immediately after it was made produced an average of 135.8 kernels per ear, compared to 146.1 kernels per ear for ears pollinated normally. Phenotypically different kernels were found on four of eight ears to which the mixture was applied. If as little as 5 min. elapsed between the time of preparing the DNA/pollen mixture and placing it on the silks, seed set was drastically reduced and no variant kernels were obtained. About 3.2 percent of the kernels from ears which were pollinated with DNA treated pollen were phenotypically different. Among the variant kernels which germinated, none of the traits segregated as expected in the subsequent generation. Ohta also utilized a procedure whereby the DNA was applied directly to the silk followed by self-pollination. Ears receiving exogenous DNA in this manner showed greatly reduced seed set (3 kernels from 5 ears) and none of them were phenotypically different.
Sanford et al. [Biotechnology and Ecology of Pollen, pp 71.76, eds. D. L. Mulcahy, G. Bergamini, Mulcahy and E. Ottavians, Springer verlag, New York (1986)] attempted to transform wild tobacco, Nicotiana langsdorffii, by germinating its pollen in the presence of either Agrobacterium tumefaciens or Ti-plasmid DNA. The treated pollen was then used to pollinate the tobacco plants. High rates of abnormal seed development and lethality were noted in the seeds produced by this treatment. Among 800 progeny plants, none synthesized nopaline nor contained T-DNA, as would be expected of Agrobacterium or Ti-plasmid transformation.
Sanford et al. [Theor. Appl. Genet., 69: 571-574 (1985)] subsequently reported unsuccessful attempts to transfer genes via pollen in corn and tomato.
Negrutiu, et al. [Biotechnology and Ecology of Pollen, pp 65-69, eds. D. L. Mulcahy, G. Bergamini, Mulcahy and E. Ottavians, Springer Verlag, New York (1986)] disclose efforts to introduce a plasmid encoding kanamycin resistance into mature germinating tobacco pollen. Tobacco pollen was germinated in the presence of the vector DNA, and various procedures, including heat shock, polyethylene glycol, and electroporation, were employed to induce uptake. Following pollination with the treated pollen, the resulting seeds were collected, germinated and screened for kanamycin resistance. A total of 400,000 seeds were screened but no resistant seedlings were found.
Fromm et al. [Nature, 319: 791-793, (1986)] disclose a plasmid comprising the cauliflower mosaic virus 35S promoter, the gene for neomycin phosphotransferase II (npt-II), and the nopaline synthase 3' region and parts of pBR322 and the use of this plasmid to show that it was transformed by electroporation into maize protoplasts from which arose stably-transformed kanamycin-resistant maize cells. That the exogenous DNA was in the maize cells was demonstrated by hybridizing an appropriate probe to the DNA of the transformed cells.
At this time there exists a clear need for an effective, efficient transformation process which can be used to incorporate exogenous DNA into flowering plants which results in functional expression of the DNA. A process appropriate for monocotyledonous crop plants would provide an opportunity to introduce important additional traits into such crops as corn, wheat, or rice.